Inflammation marks all stages of atherogenesis. DNA hypermethylation in whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aim to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low density lipoprotein receptor knockout (Ldlr-/-) mice. Ldlr-/- mice were fed an atherogenic diet and administrated with saline or 5-aza-dC (0.25mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr-/- mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had down-regulated expression of genes involved in inflammation (tumor necrosis factor α, interleukin 6, interleukin 1β and inducible nitric oxidase) and chemotaxis (CD62/L-Selectin, chemokine C-C motif ligand 2/MCP-1 (CCL2/MCP-1), CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum (ER) stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to over-expression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis.